M.M. Oliveira et al. / Journal of Molecular Structure 1215 (2020) 128291
3
2
.4. NMR and GC-MS
1.57e1605.72
m
M for CPZ and 1.27e1298.50
m
M for EtBr. The trays
ꢁ
were incubated at 37 C for 24 h and bacterial growth was revealed
by staining with resazurin.
The chemical reagents were from Sigma-Aldrich. 1H and 13C
NMR spectra were obtained using a Bruker Spectrometer, model
Avance DPX - 300 and model Avance DRX-500 operating at a fre-
quency of 300 MHz and 500 MHz for hydrogen, 75 MHz and
In order to evaluate the potential inhibition of the efflux pump
by DB Thiophene, a comparative study was made assessing its
ability to decrease the MIC antibiotics and EtBr (substrate for MDR
pumps including NorA [11,13,26,27] and MepA [15,28]), by
comparing with CPZ and CCCP which are standard EPI’s [29e32].
Eppendorf’s were prepared with the EPI’s and DB Thiophene at a
sub-inhibitory concentration (MIC/8), which corresponds to
1
CDCl
25 MHz for carbon respectively. The spectra were measured in
ꢁ
3
solvents at 27 C and chemical shifts are reported as
d
values
in parts per million (ppm) relative to tetramethylsilane (
d
0.00) as
the internal standard. The mass spectra were obtained with a Shi-
madzu QP201 GC-MS (Gas Chromatography coupled to Mass
Spectrometry) using RTX-5MS capillary column (30.0 m ꢂ 0.25 mm
x 0.30 mm) for compounds within the literature record.
520.32
for CPZ. Then 100
a 96-well microtiter tray with two-fold serial dilution by adding
00 L of antibiotics and EtBr with a final concentration ranging
from 1.27 to 1298.50 M for EtBr, 1.56e1603.36 M for norfloxacin
M for ciprofloxacin. The trays were incubated
mM for DB Thiophene, 625.56
mM for CCCP and 401.43 mM
mL of the Eppendorf’s content were transferred to
1
m
2.5. Computational details
m
m
and 1.50e1545.24
for 24 h at 37 C and bacterial growth was revealed by staining with
resazurin.
m
ꢁ
Density Functional Theory (DFT) calculations [20] were per-
formed using the Gaussian 09 program [21]. Initial coordinates
were generated according to the NMR results. The B3LYP exchange-
correlation functional [22] was applied, combined with the 6-31G*
basis set. All geometries were fully optimised within the Berny al-
gorithm, using redundant internal coordinates and considering the
Gaussian default convergence criteria. Vibrational wavenumber
calculations were carried out for the optimised geometries, at the
same theory level, to verify convergence to a real minimum within
the potential energy surface (no negative eigenvalues) and to assist
in the vibrational mode description.
2.7. Statistical analysis of the microbiological results
Antibacterial assays were performed in triplicate and results
were expressed as an average of the replicates. The results of the
tests are expressed as the geometric mean. Statistical hypothesis
analysis was applied using a Two-Way ANOVA followed by the
Bonferroni post hoc test (using the GraphPad Prism 5.0 [33]
software).
For an accurate comparison between the calculated and exper-
imental vibrational wavenumbers, the former were corrected for
anharmonicity and incomplete electron correlation treatment, us-
ing the scaling factors of 0.960 [23] and 0.942 for predicted values
below and above 2000 cm , respectively. Vibrational modes were
analysed, in terms of their Potential Energy Distribution (PED),
using the VEDA [24] program with default optimisation options.
2.8. Docking
The NorA sequence of S. aureus 1199 strain (Entry Q03325) was
retrieved from the Universal Protein Resource database (Uniprot).
The web-based SWISS-MODEL [34] service was used to build a
homology model of NorA. The HHblits-based automated mode was
used, resulting in 50 different templates, the best one being based
on the structure of the E. coli YajR transporter (PDB-ID: 3wdo).
The grid box for the docking procedure was defined as an
80Åx80Åx80 Å box around the geometrical center of the NorA
model. Partial Gasteiger charges were added to ligand atoms while
non-polar hydrogen atoms were mixed and rotational bonds
determined. Docking studies were carried out using the Lamarckian
genetic algorithm in Autodock 4.0 [35]. All other parameters were
kept at their default values. The ten best results were chosen by
least binding energy.
ꢀ
1
2.6. Evaluation of efflux pump inhibition by MIC reduction
The evaluation of the efflux pump inhibition was performed
according to Tintino et al. [12]. The two strains of S. aureus used
were: SA-1199 B, which overexpresses the NorA gene encoding the
NorA efflux protein and the multi-drug resistant (MDR) mutant
strain SA-K2068 which presents the MepA efflux pump. The strains
were maintained on blood agar base slants and, prior to use, the
ꢁ
cells were grown overnight at 37 C in Heart Infusion Agar slants.
Chlorpromazine (CPZ), carbonyl cyanide m-chlorophenylhy-
drazone (CCCP) and ethidium bromide (EtBr) were used for this
essay. Norfloxacin and Ciprofloxacin were the antibiotics used for
NorA and MepA efflux inhibition respectively. The antibiotics and
DB Thiophene were dissolved in dimethyl sulfoxide aqueous solu-
tion (5.1%, v/v). The CPZ and EtBr were dissolved in sterile water
3. Results and discussion
3.1. Structural and conformational analysis
Fig.1a and b and displays the H NMR and 13C NMR spectra of DB
Thiophene, respectively. The NMR profile (Fig. 1a) shows two
doublets, one at 6.89 ppm, assigned to H17 and H18 (J ¼ 15.6 Hz),
and another at 7.92 ppm, due to H19 and H20 (J ¼ 15.6 Hz). Their
coupling constants confirm the stereochemistry E of the double
bond. The signals at 7.48 (d, J ¼ 5.1 Hz), 7.40 (d, J ¼ 3.6 Hz) and
7.15 ppm (m) are ascribed to the hydrogens of the thiophene ring. In
1
and CCCP in methanol/water (1:1, v/v). All the compounds were
ꢁ
stored at ꢀ20 C, at a final concentration of 4162.60
m
M for DB
M for CPZ, 2597.00
for norfloxacin and 3090.48 mM for
Thiophene, 5004.50
for EtBr, 3206.71
ciprofloxacin.
m
M for CCCP, 3211.44
m
mM
mM
The Minimal Inhibitory Concentration (MIC) is defined as the
lowest concentration in which no growth can be observed [25]. The
MIC’s of CPZ, CCCP, EtBr and DB Thiophene were obtained in a
13
the C spectra the signal from the unsaturated carbonyl is at
187.7 ppm, while the ketone peak is seen at 203.8 ppm. However,
the presence of unsaturation causes a shift of the latter to high field.
The charge delocalisation is either caused by the aromatic ring
(thiophene) or by the double bond, rendering the carbonylic carbon
less electron deficient. The olefinic carbons C3 and C4 are observed
at 124.4 ppm, while C5 and C6 appear at 131.8 ppm. The carbon
atoms belonging to the thiophene rings are at 140.3, 135.6, 128.8
and 128.4 ppm. The chromatogram (Fig. S1) confirms the presence
microdilution assay using 150
solution, corresponding to a final concentration of 10 colony-
forming units/mL, followed by addition of 1350 L of brain heart
infusion (BHI) broth. Aliquots of 100 L were transferred to a 96-
well multiplate with two-fold serial dilutions by adding 100 L of
each compound solutions with final concentrations ranging from
.03 to 2081.00 M for DB Thiophene, 2.44e2502.25 M for CCCP,
mL of each strain suspended in saline
5
m
m
m
2
m
m
Amado, Ana M.
