Aminophenyl chalcones potentiating antibiotic activity and inhibiting bacterial efflux pump

Article abstract of DOI:10.1016/j.ejps.2020.105695

Chalcones and their derivatives are substances of great interest for medicinal chemistry due to their antibacterial activities. As the bacterial resistance to clinically available antibiotics has become a worldwide public health problem, it is essential to search for compounds capable of reverting the bacterial resistance. As a possibility, the chalcone class could be an interesting answer to this problem. The chalcones (2E)-1-(4′-aminophenyl)-3-(phenyl)?prop-2-en-1-one (APCHAL), and (2E)-1-(4′-aminophenyl)-3-(4-chlorophenyl)?prop-2-en-1-one (ACLOPHENYL) were synthesized by the Claisen-Schmidt condensation and characterized by ¹H and ¹³C nuclear magnetic resonance (NMR), Fourier-transform infrared (FT-IR), and mass spectrometry (MS), In addition, microbiological tests were performed to investigate the antibacterial activity, modulatory potential, and efflux pump inhibition against Staphylococcus aureus (S. aureus) multi-resistant strains. Regarding the S. aureus Gram-positive model, the APCHAL presented synergism with gentamicin and antagonism with penicillin. APCHAL reduced the Minimum inhibitory concentration (MIC) of gentamicin by almost 70%. When comparing the effects of the antibiotic modifying activity of ACLOPHENYL and APCHAL, a loss of synergism is noted with gentamicin due to the addition of a chlorine to the substance structure. For Escherichia coli (E. coli) a total lack of effect, synergistic or antagonistic, was observed between ACLOPHENYL and the antibiotics. In the evaluation of inhibition of the efflux pump, both chalcones presented a synergistic effect with norfloxacin and ciprofloxacin against S. aureus, although the effect is much less pronounced with ACLOPHENYL. The effect of APCHAL is particularly notable against the K2068 (MepA overexpresser) strain, with synergistic effects with both ciprofloxacin and ethidium bromide. The docking results also show that both compounds bind to roughly the same region of the binding site of 1199B (NorA overexpresser), and that this region overlaps with the preferred binding region of norfloxacin. The APCHAL chalcone may contribute to the prevention or treatment of infectious diseases caused by multidrug-resistant S. aureus.

Full text of DOI:10.1016/j.ejps.2020.105695

European Journal of Pharmaceutical Sciences 158 (2021) 105695
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Aminophenyl chalcones potentiating antibiotic activity and inhibiting
bacterial efflux pump
a
a,c
Marina Micaele Rodrigues Siqueira , Paulo de Tarso Cavalcante Freire
,
a
a
b
Beatriz Gonçalves Cruz , Thiago Sampaio de Freitas , Paulo Nogueira Bandeira ,
H ´e lcio Silva dos Santosa , Carlos Emidío Sampaio Nogueira
,
b
a,d
,
a,
d
a
Alexandre Magno Rodrigues Teixeira , Raimundo Luiz Silva Pereira ,
Jayze da Cunha Xavier , F a´ bia Ferreira Campina , Cristina Rodrigues dos Santos Barbosa ,
a
a
a
a
a
e
Jos ´e Bezerra de Araújo Neto , Maria Milene Costa da Silva , Jos ´e Pinto Siqueira-Júnior ,
a,*
Henrique Douglas Melo Coutinho
a
Department of Biological Chemistry, Regional University of Cariri, Crato, CE, Brazil
Science and Technology Centre - Course of Chemistry, State University Vale do Acaraú, Sobral, CE, Brazil
Department of Physics, Federal University of Cear a´ , Fortaleza, CE, Brazil
b
c
d
e
Department of Physics, Regional University of Cariri, Juazeiro do Norte, CE, Brazil
Department of Molecular Biology, Federal University of Paraíba, Jo a˜ o Pessoa, PB, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chalcones and their derivatives are substances of great interest for medicinal chemistry due to their antibacterial
activities. As the bacterial resistance to clinically available antibiotics has become a worldwide public health
problem, it is essential to search for compounds capable of reverting the bacterial resistance. As a possibility, the
Antimicrobial activity
Antibiotic potentiating activity
Efflux pump
′
chalcone class could be an interesting answer to this problem. The chalcones (2E)-1-(4 -aminophenyl)-3-(phe-
Docking
′
nyl)‑prop-2-en-1-one (APCHAL), and (2E)-1-(4 -aminophenyl)-3-(4-chlorophenyl)‑prop-2-en-1-one (ACLO-
PHENYL) were synthesized by the Claisen-Schmidt condensation and characterized by H and 13C nuclear
magnetic resonance (NMR), Fourier-transform infrared (FT-IR), and mass spectrometry (MS), In addition,
microbiological tests were performed to investigate the antibacterial activity, modulatory potential, and efflux
pump inhibition against Staphylococcus aureus (S. aureus) multi-resistant strains. Regarding the S. aureus Gram-
positive model, the APCHAL presented synergism with gentamicin and antagonism with penicillin. APCHAL
reduced the Minimum inhibitory concentration (MIC) of gentamicin by almost 70%. When comparing the effects
of the antibiotic modifying activity of ACLOPHENYL and APCHAL, a loss of synergism is noted with gentamicin
due to the addition of a chlorine to the substance structure. For Escherichia coli (E. coli) a total lack of effect,
synergistic or antagonistic, was observed between ACLOPHENYL and the antibiotics. In the evaluation of inhi-
bition of the efflux pump, both chalcones presented a synergistic effect with norfloxacin and ciprofloxacin
against S. aureus, although the effect is much less pronounced with ACLOPHENYL. The effect of APCHAL is
particularly notable against the K2068 (MepA overexpresser) strain, with synergistic effects with both cipro-
floxacin and ethidium bromide. The docking results also show that both compounds bind to roughly the same
region of the binding site of 1199B (NorA overexpresser), and that this region overlaps with the preferred binding
region of norfloxacin. The APCHAL chalcone may contribute to the prevention or treatment of infectious diseases
caused by multidrug-resistant S. aureus.
1
1
. Introduction
of active compounds and in the biosynthesis of flavonoids. Their name
stems from the Greek chalcos, meaning bronze. Chalcones have in their
–
Chalcones are used as precursors and intermediates in the synthesis
molecular structure an enone chain (Cβ=C
α
-C
–
O) with a characteristic
*
Corresponding author.
https://doi.org/10.1016/j.ejps.2020.105695
Received 9 July 2020; Received in revised form 2 December 2020; Accepted 22 December 2020
Available online 29 December 2020
0
928-0987/© 2020 Elsevier B.V. All rights reserved.

M.M.R. Siqueira et al.
European Journal of Pharmaceutical Sciences 158 (2021) 105695
α
,β-unsaturated carbonyl (Mari n˜ o et al., 2016). This compound class has
chlorophenyl)‑prop-2-en-1-one (C15H12NOCl, hereafter named ACLO-
a yellow coloration; however, in an alkaline medium, it presents a red
color. Chalcones are present in the pigmentation of flowers, making
them attractive to birds and insects that contribute to the pollination
process of plants (Katsori and Hadjipavlou-Litina, 2009).
PHENYL). The evaluation of inhibition of the efflux pump for both
compounds was also performed. Two strains of S. aureus were used:
1199B, which overexpresses the NorA gene and the multi-drug resistant
(MDR) mutant strain K2068, which presents the MepA efflux pump.
The synthesis of chalcone compounds is essential, given that the
volume of material that can be extracted through natural processes is
usually insignificant and inappropriate for use at an industrial scale.
Among the methods of chalcones syntheses, the aldol condensation of
Claisen-Schmidt stands out (Nandedkar et al., 2013). Recently, studies
have shown different pharmacological activities for chalcones, such as
antifungal (Gupta and Jain, 2015), antibacterial (Tran et al., 2012),
anticancer (Bandeira et al., 2019; Bhat et al., 2005; Katsori and Hadji-
pavlou-Litina, 2009), antioxidant, and antidiabetic activity (Eichen-
berger et al., 2017).
2. Materials and methods
2.1. Synthesis of chalcones
The p-aminochalcones derivatives used in this study were synthe-
sized via Claisen-Schmidt condensation conducted under basic condi-
tions. A solution of p-aminoacetophenone (2 mmol) in ethanol (5 mL)
was added to a solution of benzaldehyde (2 mmol) in ethanol (5 mL)
containing 10 drops of 50% v/v sodium hydroxide, and the resulting
mixture was stirred for 48 h. The mixture was filtered under vacuum,
washed with cold water to pH 7.0, and analyzed by Thin-layer chro-
matography (TLC) (Scheme 1).
Health care-associated infections (HCAI) are considered a major
problem for public health, impacting on morbidity and mortality rates
during the hospitalization period, as well as increasing diagnostic and
therapeutic expenses (O’Neill, 2016; Oliveira et al., 2010). Antibiotic
resistance is considered a world health disorder, which impacts the
effectiveness of antibiotics for the treatment of infections (WHO, 2009).
In this context, the use of a natural or synthetic substance to modulate
bacterial resistance against an antibiotic has proven to be an alternative
to drugs that are more effective in the treatment of infectious diseases
′
Some characteristics of (2E)ꢀ 1-(4 -aminophenyl)ꢀ 3-(phenyl)‑prop-
2-en-1-one (APCHAL) are given as follows. Yellow solid (Yield:
◦
ꢀ 1
cm): 3522, 3434, 1623,
25.60%), m.p. 109.3 – 109.9 C; FT-IR (KBr,
ν
1
1578, 1554. H NMR (CD
3
OD, 300 MHz) δ: 7.40 – 7.42 (m, H-3/H-5, H-
′
′
′
′
4), 7.93 (d, H-2 /H-6 , J = 8.7 Hz), 6.73 (d, H-3 /H-5 , J = 8.7 Hz), 7.69 –
1
3
7.72 (m, H-2/H-6, H-
α, H-β). C NMR (CD
3
′
OD, 75 MHz) δ: C-1 136.8, C-
′
′
′
(
Rios et al., 1988).
2/C-6 129.6 C-3/C-5 130.1, C-4 131.4, C-1 128.2, C-2 /C-6 132.6, C-3 /
′
′
–
–
Bacterial resistance to antibiotics can be internal or obtained through
C-5 115.1, C-4 154.8, C-
α
123.3, C-β 144.4, C O 190.3. MS (EI) m/z
13NO/223 (Bandeira et al., 2019; Santiago
+
.
genetic transmission or mutation, modifying the mechanisms by which
antibiotics become incapable of combating bacterial strains (Sun et al.,
(M 223), calcd for C15
H
et al., 2018).
′
2
014). An important mechanism of bacterial resistance is the presence of
Some characteristics of (2E)ꢀ 1-(4 -aminophenyl)ꢀ 3-(4-clor-
ophenyl)‑prop-2-en-1-one (ACLOPHENYL) are given as follows. Yellow
proteins from efflux pumps in their membranes. It is known that the
efflux pump is one of the main causes of drug resistance (Webber and
Piddock, 2003). The efflux pumps act by active transport, causing the
extrusion of one or several types of antibiotics from the bacterial cyto-
plasm (Sharma et al., 2019). As examples of multi-drug resistance
pumps (MDR pumps) are NorA and MepA, analyzed in this article. In this
way, efflux pump inhibitors become an important therapeutic alterna-
tive in the treatment of infectious diseases.
◦
ꢀ 1
solid (Yield: 53.59%), m.p. 162,9 - 163,3 C; FT-IR (KBr,
ν
cm): 3555,
1
3350, 1621, 1570, 1550, 1490. H NMR (CD
3
OD, 300 MHz): δ 7.91 (d,
′
H-2/H-6, J = 8.73 Hz), 7.69 (d, H-3/H-5, J = 8.79 Hz), 7.42 (d, H-2 /H-
′
′
3
′
6 , J = 8.46 Hz), 6.68 (d, H-3 /H-5 , J Hz), 7.70 (d, H-
α
, J = 14.28 Hz),
1
7.74 (d, H-β, J = 15.60 Hz). C NMR (CD
3
OD, 75 MHz): δ C-1 135.6, C-
′
′
′
2/C-6 132.7, C-3/C-5 130.9, C-4 137.5, C-1 127.6, C-2 /C-6 130.8, C-
′
′
′
–
–
O 189.9. MS (EI) m/z
3 /C-5 114.6, C-4 155.8, C-
α
124.3, C-β 142.9, C
+
.
Previous studies had reported that some compounds can inhibit
efflux pumps, such as certain compounds derived from indole (Lepri
et al., 2016). Derivatives of 2-arylquinoline (Felicetti et al., 2020) and
boronic acid derivatives have the ability to inhibit NorA efflux pumps
(M 257.5), calcd for: C15H12NOCl/257.5 (Bandeira et al., 2019).
2.2. Microorganisms
(
Fontaine et al., 2014), while compounds derived from 2-phenylquino-
In the studies related to the modification of antibiotic activity, the
standard bacterial strains used were Escherichia coli ATCC 25922 and
Staphylococcus aureus ATCC 25923, while the resistant strains were
E. coli 06 and Staphylococcus aureus 10. The E. coli 06 strain is resistant to
several β-lactams such as cephalothin, cephalexin, and ceftriaxone,
while the Staphylococcus aureus 10 strain is resistant to β-lactams, fluo-
roquinolones, and macrolides.
line showed the ability to inhibit NorA and MepA efflux pumps (Feli-
cetti et al., 2018). Previous studies had shown that chalcones can exhibit
antibacterial properties via efflux pump inhibition (Dan and Dai, 2020;
Holler et al., 2012; Thai et al., 2015). Holler et al. screened a library of
1
17 chalcones for efflux pump inhibition activity against NorA (Holler
et al., 2012). In this study, twenty chalcones were shown to be inhibitors
of the NorA efflux pump in everted membrane vesicles. It was also
shown that two chalcones exhibited potential activity equal to the
known efflux pump inhibitor reserpine. These results lead them to
suggest that chalcones can be transformed into drugs for overcoming
multi-drug resistance based on efflux transporters of microorganisms.
Thai et al. carried out a virtual screening and molecular docking in a
series of forty-seven natural compounds, including chalcones, to inves-
tigate the novel Staphylococcus aureus NorA efflux pump inhibitors (Thai
et al., 2015). They observed that seven of them were predicted as NorA
In the efflux pump tests, the bacterial strains of Staphylococcus aureus
1199B (which overexpresses the NorA gene encoding the NorA efflux
protein) and Staphylococcus aureus K2068 (which presents the MepA
efflux pump) were used. All the bacterial strains were cultivated in heart
infusion agar (HIA) during 24 h. Brain heart infusion (BHI) broth was the
medium used at the time of the test.
2.3. Antimicrobial activity and effect
′
inhibitors, among them the (2E)ꢀ 1-(4 -aminophenyl)- 3-(4-chlor-
2.3.1. Drugs
ophenyl)‑prop-2-en-1-one, which is one of the chalcones that will be
investigated in this work.
For the evaluation of the antibiotic potentiating activity of the
APCHAL and ACLOPHENYL chalcones, two antibiotics were used: the
aminoglycoside antibiotic gentamicin, the β-lactam antibiotic penicillin.
In tests with efflux pumps, the antibiotics chosen act as specific
substrates for each one of the bacterial strains: norfloxacin for SA 1199B,
and ciprofloxacin for SA K2068.
An attempt to contain the bacterial resistance by efflux pump inhi-
bition happens through the association of antibiotics with substances
that are capable of inhibiting these pumps. In this study, we investigated
the antimicrobial and antibiotic potentiating activity of the chalcones
′
(
2E)ꢀ 1-(4 -aminophenyl)ꢀ 3-(phenyl)‑prop-2-en-1-one
(C15
H
13NO,
The solutions were prepared on the basis of recommendations
established in CLSI (CLSI, 2008) and diluted in sterile water to reach a
′
hereafter named APCHAL) and (2E)ꢀ 1-(4 -aminophenyl)ꢀ 3-(4-
2

M.M.R. Siqueira et al.
European Journal of Pharmaceutical Sciences 158 (2021) 105695
concentration of 1024 µg/mL. In addition to antibiotics, ethidium bro-
mide was used to check for efflux pumps.
Universal Protein Resource database (Uniprot, Entry Q03325). The web-
based SWISS-MODEL service was used to build a homology model of
NorA (Waterhouse et al., 2018). The HHblits-based automated mode
was used, resulting in 50 different templates with the best one based on
the structure of E. coli YajR transporter (PDB-ID: 3wdo).
For the tests, a solution prepared from the APCHAL and ACLO-
PHENYL compounds were used at a concentration of 10 mg/mL, dis-
solved in 0,5 mL DMSO (dimethyl sulfoxide), and further diluted with
distilled water to a concentration of 1024 µg/mL.
The MepA model was generated by retrieving the protein sequence
for the NCTC 8325 strain from the Uniprot database. The same pro-
cedure as before was utilized. The template of the multi-drug and toxic
compound extrusion (MATE) transporter of the Bacillus halodurans
(PDB-ID: 5c6n) was chosen for the homology model.
2
.3.2. Antibacterial activity
MIC (minimum inhibitory concentration) was determined using
broth microdilution tests (Coutinho et al., 2008) with 96-well plates.
The bacteria were suspended in BHI broth at a concentration of 105
CFU/mL. The final concentration of the APCHAL and ACLOPHENYL
Both models were then uploaded to the Molecular Dynamics on Web
(MDWeb) (Hospital et al., 2012) service, where they were solvated and
submitted to a 5 ps molecular dynamics run in canonical ensemble
(NVT), followed by a 0.5 ns production run in isothermal–isobaric (NPT)
ensemble using the AMBER99 force field. The root mean square devia-
tion (RMSD) of both models reached a plateau after approximately 0.4
ns.
samples ranged from 512 to 8 g/mL. MICs were evidenced using a
μ
colorimetric method with resazurin blue, and their values correspond to
the lowest concentrations capable of inhibiting bacterial growth.
2
.3.3. Modulation
To evaluate the APCHAL substance as a microbial resistance modu-
The grid box for the docking procedure was defined as a 70 Å x 70 Å
x 70 Å box around the geometrical center of the protein model. Partial
Gasteiger charges were added to ligand atoms, non-polar hydrogen
atoms were merged, and rotational bonds were determined. Docking
studies were carried out through the Lamarckian genetic algorithm in
Autodock 4.0 (Morris et al., 2009), and all parameters were kept at their
default values. The ten best results were chosen by the lowest binding
energy (kcal/mol).
lator, the MIC of antimicrobials (gentamycin, and penicillin) was
determined in the presence of the compound in a sub-inhibitory con-
centration (MIC/8), based on the methodology by Coutinho et al.
2008). Microorganisms at the 105 CFU/mL concentration were sus-
(
pended in the BHI medium in 96-well microdilution plates. The plates
◦
were incubated at 37 C for 24 h, after which time readings were per-
formed using resazurin.
2
.3.4. Efflux pump
2.6. Data analysis
The evaluation of inhibition of the efflux pump was performed ac-
cording to Tintino et al. (2016). Two strains of S. aureus were used:
S. aureus 1199B (SA-1199B), which overexpresses the NorA gene
encoding the NorA efflux protein, and the multi-drug resistant (MDR)
mutant strain S. aureus (SA-K2068) which presents the MepA efflux
pump. The strains were maintained on blood agar base slants, and,
The tests were performed in triplicates, and the results were
expressed as the geometric mean. A two-way NOVA with Tukey’s post-
hoc Bonferroni test statistical analysis was applied to verify the anti-
biotic potentiating activity of the APCHAL using the program Graph-
PadPrism 6.02 [30][30], where p < 0.05 was considered significant.
◦
before use, the cells were grown overnight at 37 C in Heart Infusion
Agar slants. The chlorpromazine, carbonyl cyanide m-chlorophenylhy-
drazone (CCCP), and ethidium bromide were obtained from Sigma
Aldrich Co. Ltd. Norfloxacin and ciprofloxacin were the antibiotics used
in NorA and MepA efflux inhibition, respectively. The antibiotics and the
compounds under study were first dissolved in dimethyl sulfoxide
3. Results and discussion
3.1. Structural determination
The 13C NMR spectra of the APCHAL and ACLOPHENYL chalcones
–
(
DMSO). Subsequently, the compounds dissolved in DMSO underwent a
presented characteristic signals of ketone (C
–
O) conjugated to the
α
,
new dilution, this time in sterile water. Chlorpromazine and ethidium
β-unsaturated system with values of δ 190.3 and 189.9 ppm, respec-
C
bromide were dissolved in sterile water and CCCP in methanol/water
tively. They also revealed signs for C
α with values of δ 123.3 and 124.3
C
◦
(
1:1, v/v). All the compounds were stored at ꢀ 20 C at a final concen-
ppm and C with values of δ 144.4 and 142.9 ppm for APCHAL and
β
C
1
tration of 1024 µg/mL. To evaluate the inhibition capacity of the efflux
pumps, the MICs of the strain-specific antibiotics, as well as the ethidium
bromide used as control, were compared with the MICs of their associ-
ations with standard inhibitors.
ACLOPHENYL, respectively, characteristic of enones. In addition, the H
signals in C-sp2 were observed, forming a doublet with coupling values
of 12.12 - 15.96 Hz for the H
α
β
and H characteristics of trans hydrogens.
This information justifies the prediction for the synthesis of chalcones in
–
O (Bandeira
The test solutions used in the aforementioned controls were prepared
in Eppendorf microtubes containing medium and inoculum. The test
solutions used for comparison were added to the chalcones and standard
inhibitors in quantities that correspond to MIC/8 (subinhibitory con-
centration). Then 100µL of Eppendorf’s content were transferred to 96-
well microtiter tray with two-fold serial dilution by adding 100µL of
antibiotics and ethidium bromide with final concentration ranging from
the generation of the double bond conjugated with C
–
et al., 2019). The molecular structures of the chalcones APCHAL and
1
13
ACLOPHENYL are shown in Fig. 1, and the H and C NMR spectra are
given in the supplemental material.
3.2. Antibiotic activity study
0
.5 to 512 µg/mL. The trays were incubated at 37 ◦C for 24 h, and
By one hand, the MICs obtained for APCHAL with standard and
bacterial growth was revealed by staining with resazurin.
resistant bacterial strains of Staphylococcus aureus (SA) and E. coli (EC)
were: 1024
mL with EC ATCC and 1024
MICs obtained for ACLOPHENYL with standard and resistant bacterial
strains were: 512 g/mL with SA ATCC and 1024 g/mL with SA10;
1024 g/mL with EC ATCC and 1024 g/mL with EC 06. The subin-
μ
g/mL with SA ATCC and 1024
μ
g/mL with SA 10; 1024 g/
μ
2
.4. Octanol-water partition coefficient calculation
μ
g/mL with EC 06. By the other hand, the
The Octanol-water partition coefficient was calculated using
μ
μ
Molinspiration molecular property calculation services (Molinspiration,
μ
μ
2
020).
hibitory concentration for APCHAL chalcone was 128 µg/mL for all
tested strains, corresponding to 188 µL of volume of the compound in the
Eppendorf microtube. The ACLOPHENYL had a subinhibitory concen-
tration of 64 µg/mL for SA ATCC, which represents 94 µL of the volume
of this compound in the Eppendorf microtube. For the other strains, the
2
.5. Docking studies
NorA sequence of S. aureus 1199B strain was retrieved from the
3

M.M.R. Siqueira et al.
European Journal of Pharmaceutical Sciences 158 (2021) 105695
Fig. 1. Molecular structures of the APCHAL and ACLOPHENYL synthetic chalcones.
ACLOPHENYL follows the same pattern of the APCHAL.
receptor in the same way (Silverman, 2004).
Figs. 2 and 3 show, respectively, the MIC of the antibiotics genta-
micin and penicillin in the presence and absence of the APCHAL and
ACLOPHENYL chalcones.
A possible explanation for the loss of synergism is that the chlorine
addition in ACLOPHENYL reduces its membrane permeability. Studies
have shown that there is a good correlation between physicochemical
properties, such as octanol/water partitioning coefficients and mem-
brane permeability (Lipinski et al., 1997). Drugs with an octanol/water
partitioning coefficient (logP) close to 2 are generally readily adsorbed
while compounds with logP > 4 have lower permeability (Artursson
et al., 2001). The logP for APCHAL and ACLOPHENYL are, respectively,
2.89 and 3.56. In other words, the chlorine addition slightly reduces the
membrane permeability and may explain the difference in the syner-
gistic effect.
Two different antibiotic classes were used to test the modulation
effect: gentamicin, an aminoglycoside, and penicillin, a beta-lactam. The
effect was tested in two models using Gram-positive and Gram-negative
bacteria. Regarding the Staphylococcus aureus Gram-positive model, the
APCHAL chalcone presented synergy with the gentamicin and antago-
nism with penicillin. APCHAL reduced the MIC of gentamicin by almost
7
0%. Gentamicin is an antibiotic that acts by inhibiting intracellular
protein production (Cushnie et al., 2016). Penicillin, as a β-lactam, has a
different mechanism of action. It acts outside the cell, preventing the
formation of cross-bridge in the cell wall (Kapoor et al., 2017). This fact
may explain the difference in the synergistic pattern found. Differently,
on the E. coli Gram-negative model, APCHAL exhibits an antagonism to
aminoglycosides and indifference to β-lactams. The loss of effectiveness
in this bacterial model may be explained both by structural differences
between Gram-positive and Gram-negative bacteria, which alter
permeability and by drug interaction with their targets (Zengin and
Baysal, 2014).
The evaluation of inhibition of the efflux pump for both compounds
was performed against two strains of S. aureus: SA-1199B, which over-
expresses the NorA gene encoding the NorA efflux protein (Fig. 4) and
the multi-drug resistant (MDR) mutant strain SA-K2068, which presents
the MepA efflux pump (Fig. 5).
Ethidium bromide (EB) is a dye that presents antibiotic activity due
to its characteristic of DNA intercalating, promoting damage to the DNA
structure, causing cell death (Shabestari et al., 2017). The only mecha-
nism used by bacteria to resist the action of the ethidium bromide is the
efflux pump (Pal et al., 2020). Its use associated with standard efflux
pump inhibitors is a technique used in several studies to investigate the
presence or absence of efflux pumps (Limaverde et al., 2017; Tintino
et al., 2018, 2016).
When comparing the effects of antibiotic-modifying activity between
the two substances tested, ACLOPHENYL and APCHAL, a loss of syner-
gistic effect is noted with gentamicin. This is due to the addition of a
chlorine to the structure of the substance (APCHAL to ACLOPHENYL) in
the Gram-positive model, while in penicillin the results were indifferent
or antagonistic. For E. coli, there was a total lack of effect, synergistic or
antagonistic, between ACLOPHENYL and antibiotics. The structure of
ACLOPHENYL, when compared to APCHAL, differs only by a chlorine
ligand in position 4 of the chalcones. Nevertheless, this single molecular
change was able to reverse the synergistic effect that is observed with
APCHAL when associated with gentamicin. In addition, compounds that
exhibit structural similarity do not necessarily bind to the target
In our tests, we utilized two standard efflux pump inhibitors, the
CCCP and the chlorpromazine, each with its own inhibition mechanism.
The CCCP acts by causing a disturbance in the electrochemical potential
due to a decrease in the production of adenosine triphosphate (ATP)
leading to efflux pump inhibition. Whereas the chlorpromazine acts via
competitive inhibition on antibiotics, interacting directly with the efflux
pumps and inhibiting them [25,42]. Thus, the presence of the efflux
pump will be visualized when the minimum inhibitory concentration of
ethidium bromide associated with a standard inhibitor is lower than the
MIC of the bromide control, indicating the inability of the bacterial cell
to use the efflux mechanism.
We can evidence the presence of efflux pumps in strains 1199B and
K2068. In strain 1199B, both chlorpromazine and CCCP associated with
bromide showed MICs that were lower than the control, showing the
presence of efflux pumps with distinct mechanisms of the expulsion of
intracellular bromide. In strain K2068, we have efflux pumps sensitive
only to CCCP in the presence of ethidium bromide, showing that a likely
competitive inhibition mechanism is not effective for this bacterium in
relation to ethidium bromide. This fact leads us to infer that the most
effective mode of action by which the two chalcones inhibit the NorA
and MepA efflux pumps is related to the inhibition of the generator
mechanisms of the motive force that acts as a propellant of the respec-
tive pumps. These results were corroborated by the study conducted by
Gupta et al. [42] who showed that the IMRG4 synthetic chalcone has
antibacterial activity against several multi-resistant strains of Staphylo-
coccus aureus. Gupta et al. [42] also showed that IMRG4 inhibits the
NorA efflux pump by interference in the proton motive force, preventing
the generation of energy necessary for the pump operation (Gupta et al.,
Fig. 2. Minimum inhibitory concentration (MIC) of the antibiotics gentamicin
and penicillin in the presence and absence of the chalcone APCHAL towards
S. aureus 10 and E. coli 06.
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M.M.R. Siqueira et al.
European Journal of Pharmaceutical Sciences 158 (2021) 105695
Fig. 3. Minimum inhibitory concentration (MIC) of the antibiotics gentamicin and penicillin in the presence and absence of the chalcone ACLOPHENYL towards
S. aureus 10 and E. coli 06.
Fig. 4. MIC of norfloxacin and ethidium bromide alone and in association with the standard inhibitors and chalcones APCHAL and ACLOPHENY against the strain
S. aureus 1199B
Fig. 5. MIC of ciprofloxacin and ethidium bromide alone and in association with the standard inhibitors and chalcones APCHAL and ACLOPHENY against the strain
S. aureus K2068.
5

M.M.R. Siqueira et al.
European Journal of Pharmaceutical Sciences 158 (2021) 105695
2
019).
The standard inhibitors revealed the presence of efflux pumps sen-
Fig. 7 displays the best poses of APCHAL (pink) and ACLOPHENYL (light
green), and the best pose of ciprofloxacin (light blue) on the binding site
of the NorA model. 2D ligand-protein interaction diagrams along with
contact distances for both molecules are available as Figures S7 and S8
of the Supplementary Material. The phenyl and chlorophenyl rings are
sitive to both inhibition mechanisms, both in strain 1199B with relation
to norfloxacin and in strain K2068 in relation to ciprofloxacin. As can be
seen from Figs. 4 and 5, both chalcones presented a synergistic effect
with norfloxacin and ciprofloxacin against multiple drug-resistant
Staphylococcus aureus, although the effect is much less pronounced
with ACLOPHENYL. The synergistic effect of the APCHAL is particularly
notable against the strain K2068. The less pronounced synergistic effect
of ACLOPHENYL chalcone may be related to a lower efficacy in the main
sensitivity mechanism presented by the efflux pumps, and this lower
efficacy may be correlated to the structural difference of APCHAL and
ACLOPHENYL chalcones.
positioned to facilitate
π
-π stacking interactions with the Phe61. It is
observed that APCHAL and ACLOPHENYL molecules overlap almost
completely with ciprofloxacin, with the three molecules making close
contacts with the same residues, such as Met65, Met171, and Asn204. In
other words, the MepA model cannot capture the effect of adding a
chlorine atom. These results show that the chalcones can act as efflux
pump inhibitors, as previously proposed for other chalcones (Garcia
et al., 2020; Rezende-Júnior et al., 2020) through a combination of in-
hibition essays and docking studies.
Our docking studies show that both APCHAL and ACLOPHENYL bind
to the binding site of the NorA model, although in different regions of
the binding site. Fig. 6 shows the best poses of APCHAL (Pink) and
ACLOPHENYL (Light Green), as well as the best pose of norfloxacin
4
. Conclusions
(
Light Blue) on the binding site of the NorA model. 2D ligand-protein
The APCHAL chalcone presented a synergistic effect with gentamicin
interaction diagrams along with contact distances for both molecules
are available as Figures S5 and S6 of the Supplementary Material. Both
APCHAL’s phenyl, as well as ACLOPHENYL’s chlorophenyl, are posi-
against a multi-drug resistant Staphylococcus aureus strain. However,
when a chlorine atom was added to this structure (ACLOPHENYL), the
synergistic effect was lost. The loss of a synergistic effect was associated
with the presence of the chlorine atom in the molecular structure of the
chalcone ACLOPHENYL. However, the presence of synergism or antag-
onism depends on factors such as the location of the halogen ligand, the
types of associated antibiotic ligands to be combined, and the bacterial
model involved. Docking studies have shown that the addition of the
chlorine impairs the compound’s ability to bind to the binding site of the
efflux pump proteins, reducing the proposed ability to inhibit the pump.
However, other mechanisms of action could not be ruled out.
tioned, such as to facilitate
π
-π stacking interactions with the Phe139
residue. In addition, APCHAL makes close contacts with amino acid
residues Ser214 and Ser218 in a similar fashion as norfloxacin. As can be
seen from the figure, the best pose of APCHAL overlaps with that of
norfloxacin, while ACLOPHENYL does not. This may explain the much
greater synergistic effect of APCHAL compared to ACLOPHENYL. In
other words, the bulky and electronegative chlorine changes the binding
pose of ACLOPHENYL in a way that is less efficient in inhibiting the
efflux pump when compared to APCHAL. According to Palazzotti et al.
the description of the binding of ciprofloxacin to the orthosteric binding
site of another NorA model is roughly equivalent to ours, although the
preparation of the model is quite different (Palazzotti et al., 2019). One
should note that their residue numbering is not equal to ours, with
Phe139 appearing as Phe140 and Glu221 as Glu222 and so on. One
should also note the difference in antibiotics (norfloxacin versus cipro-
floxacin) when comparing these results.
Credit authorship contribution statement
M.M.R.S.: Investigation, and writing-original draft. H.S.S.:
Conceptualization, performed the NMR study, and writing-review and
editing. P.T.C.F.: Supervision and writing-review and editing. T.S.d.F.:
Investigation of the modulation of multidrug resistance efflux pump
activity and revised the manuscript. B.G.C.: Investigation of the anti-
microbial activity assays. J.d.C.X.; Statistical analysis. P.N.B.: Co-
supervision and performed the synthesis of the chalcone. M.M.C.d.S.:
Additionally, APCHAL and ACLOPHENYL bind to the binding site of
the MepA model in almost the same way, as can be seen from Fig. 7.
Fig. 6. The best poses of APCHAL (Pink) and ACLOPHENYL (Light Green), as well as the best pose of Norfloxacin (Light Blue) on the binding site of the NorA model.
6

M.M.R. Siqueira et al.
European Journal of Pharmaceutical Sciences 158 (2021) 105695
Fig. 7. The best poses of APCHAL (Pink) and ACLOPHENYL (Light Green), and the best pose of Ciprofloxacin (Light Blue) on the binding site of the MepA model.
Scheme 1. Preparation of chalcones a) NaOH 50% w /v, ethanol, r.t., 48 h.
Data curation. R.L.S.P.: Methodology. A.M.R.T.: Writing-review and
editing. C.R.d.S.B.: Visualization. C.E.S.N.: Software, formal analysis,
molecular docking, and writing-review and editing. J.P.S.Jr.: Project
administration. H.D.M.C.: Methodology and project administration. J.
B.d.A.N.: Resources and funding acquisition.
Funding
This work was supported by the Conselho Nacional de Desenvolvi-
mento Científico e Tecnol o´ gico – CNPq [grant number 305719/2018-1].
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
Author contributions
Investigation, and writing-original draft, M.M.R.S.
Conceptualization, performed the NMR study, and writing—review
and editing. H.S.S. supervision and revised the manuscript. P.T.C.F.
performed the efflux pumps assays, and writing—review and editing, T.
S.d.F. validation, M.M.R.S. carried out the antimicrobial activity assays,
B.G.C.
We thank the Northeastern Center for the Use and Application of
Nuclear Magnetic Resonance (CENAUREM) for the spectroscopy
measurements.
Statistical analysis, J.d.C.X. performed the synthesis of the chalcone,
P.N.B. data curation, M.M.C.d.S. methodology, R.L.S.P. funding acqui-
sition, and writing—review and editing, A.M.R.T. visualization, C.R.d.S.
B. software, formal analysis, molecular docking, and writing—review
and editing, C.E.S.N. project administration, J.P.S.Jr. methodology and
project administration H.D.M.C. resources and funding acquisition, J.B.
d.A.N.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.ejps.2020.105695.
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