Design, synthesis, antibacterial activity evaluation and molecular modeling studies of new sulfonamides containing a sulfathiazole moiety

Article abstract of DOI:10.1039/d1nj00150g

Sulfonamides represent the oldest synthetic antibacterial agents; however, their central position in controlling bacterial diseases has been seriously damaged by the development of widespread resistance. Herein, we revisited sulfathiazole, a commercial member of antibacterial sulfa drugs, intending to overcome sulfonamide resistance and identify new drug candidates through molecular modifications. We synthesized twelve sulfonamides (SA1-SA12) by replacing the amino group on the phenyl ring with various substituents and introducing a thiophene ring on the core scaffold of sulfathiazole. The obtained compounds and additionally two commercial sulfonamides, sulfathiazole and sulfadiazine, were extensively screened for their antimicrobial activities. The results indicated that new sulfonamides, unlike traditional ones, were selectively effective against various Staphylococcus aureus strains. Introducing a bulky lipophilic substituent at the para position of the phenyl ring significantly increased the antibacterial activities of the compounds against Staphylococcus aureus. The compounds demonstrating favourable selectivity indices were further evaluated for their membrane potential perturbation and DNA interaction properties. The obtained data showed that these are not supporting mechanisms for the antibacterial activities of the modified sulfathiazole derivatives. In order to rationalize the activity of the three most active compounds, SA7, SA11 and SA12, against S. aureus ATCC 25923, their binding hypotheses within the catalytic site of Staphylococcus aureus dihydropteroate synthase, the validated target enzyme of sulfonamides, were generated via molecular docking and further dissected using molecular dynamics simulations and dynamic 3D pharmacophores (dynophores).

Full text of DOI:10.1039/d1nj00150g

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Design, synthesis, antibacterial activity evaluation
and molecular modeling studies of new
sulfonamides containing a sulfathiazole moiety†
Cite this: New J. Chem., 2021,
45, 8166
ab
c
Tugba Mes-eli,
S- engul Dilem Dogan, *b Miyase Gozde Gunduz,
˘
˘
¨
¨
¨
¨
a
d
e
Zulbiye Kokbudak,
Sanja Skaro Bogojevic,
Theresa Noonan,
¨
¨
d
e
d
Sandra Vojnovic,
Gerhard Wolber
and Jasmina Nikodinovic-Runic
Sulfonamides represent the oldest synthetic antibacterial agents; however, their central position in
controlling bacterial diseases has been seriously damaged by the development of widespread resistance.
Herein, we revisited sulfathiazole, a commercial member of antibacterial sulfa drugs, intending to
overcome sulfonamide resistance and identify new drug candidates through molecular modifications.
We synthesized twelve sulfonamides (SA1–SA12) by replacing the amino group on the phenyl ring with
various substituents and introducing a thiophene ring on the core scaffold of sulfathiazole. The obtained
compounds and additionally two commercial sulfonamides, sulfathiazole and sulfadiazine, were
extensively screened for their antimicrobial activities. The results indicated that new sulfonamides, unlike
traditional ones, were selectively effective against various Staphylococcus aureus strains. Introducing a
bulky lipophilic substituent at the para position of the phenyl ring significantly increased the antibacterial
activities of the compounds against Staphylococcus aureus. The compounds demonstrating favourable
selectivity indices were further evaluated for their membrane potential perturbation and DNA interaction
properties. The obtained data showed that these are not supporting mechanisms for the antibacterial
activities of the modified sulfathiazole derivatives. In order to rationalize the activity of the three most
active compounds, SA7, SA11 and SA12, against S. aureus ATCC 25923, their binding hypotheses within
the catalytic site of Staphylococcus aureus dihydropteroate synthase, the validated target enzyme of
sulfonamides, were generated via molecular docking and further dissected using molecular dynamics
simulations and dynamic 3D pharmacophores (dynophores).
Received 10th January 2021,
Accepted 25th March 2021
DOI: 10.1039/d1nj00150g
rsc.li/njc
the discovery of novel antibacterial agents with different scaffolds
or a new mechanism of action is of utmost importance.
Introduction
Bacterial infections have been a serious threat to public health
Sulfonamide is one of the most common functionalities
over centuries causing significant morbidity and mortality in medicinal chemistry integrated into the structures of ther-
worldwide.1 Despite continuous efforts to develop and launch apeutically valuable molecules.4 In addition to being the first
new antibacterial drugs, the rate and the spread of bacterial synthetic antibacterial agent, sulfonamides also provide a wide
infections have never been stagnated.2 The primary obstacle to spectrum of biological activities such as carbonic anhydrase
bringing bacterial infections completely under control is the inhibition, insulin release induction, and anti-inflammation.5,6
emergence of multidrug-resistant bacterial strains.3 Therefore,
Antibacterial sulfonamides, such as sulfathiazole, sulfadia-
zine, and sulfisoxazole (Fig. 1), exert their mechanism of action
by interfering with the bacterial folate pathway.7
a Department of Chemistry, Faculty of Science, Erciyes University, 38039, Kayseri,
Turkey
Dihydropteroate synthase (DHPS), one of the enzymes
employed in folate biosynthesis, catalyzes the key reaction between
dihydropteroate diphosphate (DHPP) and p-aminobenzoic acid
(PABA) to form dihydropteroic acid.8 Sulfonamides target the PABA
binding site of DHPS through their similar chemical structures to
PABA and inhibit the formation of dihydropteroic acid (Fig. 2).
This interruption in the folate biosynthesis significantly sup-
b Department of Basic Sciences, Faculty of Pharmacy, Erciyes University, 38039,
Kayseri, Turkey. E-mail: dogandilem@gmail.com; Tel: +90 352 2076666-28032
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe
University, Sıhhiye, 06100, Ankara, Turkey
d Institute of Molecular Genetics and Genetic Engineering, University of Belgrade,
Vojvode Stepe 444a, 11000 Belgrade, Serbia
e Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy,
¨
¨
Freie Universitat Berlin, Konigin-Luise-Str. 2 + 4, 14195 Berlin, Germany
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1nj00150g presses bacterial growth and reproduction.9
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Fig. 1 Examples of antibacterial sulfa drugs.
Although sulfonamides were the first group of compounds
In light of these considerations, we aimed to carry out various
that acted selectively on bacteria, their clinical use is highly molecular modifications on a representative sulfa drug, sulfathia-
limited nowadays. There are some important reasons for this zole, to overcome resistance and safety problems observed with
limitation. One of them is that many other and more effective traditional sulfonamides. To the core scaffold of sulfathiazole, we
antibacterial agents were discovered after their registration in added the thiophene ring, which is one of the most common
the 1930s. The second, and maybe the most important one, is heterocycles in biologically active molecules including anti-
the rapid drug resistance that occurred against sulfonamides. bacterial agents.18,19 With this modification, we aimed to extend
Other reasons that restrict the use of sulfonamides are the severe the structure beyond the native substrate (PABA) binding pocket
toxicity and the allergic side effects that are quite common of DHPS. Additionally, we introduced diverse substituents on
among patients.11
the phenyl ring instead of the amino group, which is one of the
Sulfathiazole is a short-acting antibacterial sulfa drug. Although key moieties of classical sulfonamides (Fig. 3). To determine the
its sole use has been replaced by less toxic alternatives, it is still effects of these modifications on the antibacterial activity, we
present in commercial products in combination with other anti- tested the obtained compounds against different organisms.
bacterial agents, especially for the treatment of vaginal infections.12
Molecular modification is a frequently applied drug design
Results and discussion
Chemistry
strategy based on the chemical alteration of a lead compound.13
This approach targets the improvement of pharmacological and
pharmacokinetic profiles, such as increased potency, reduced
toxicity, and enhanced chemical properties.14 Molecular mod-
ification of existing chemical scaffolds stands also as a pro-
mising tool to overcome drug resistance observed in not
only antibacterial15 but also antitubercular and anticancer
therapies.16,17 Considering the gradually increasing antibacter-
ial resistance worldwide, inexpensive sulfonamides might be
reconsidered in the treatment of infectious diseases through
appropriate structural modifications.
The synthetic route employed to produce the target sulfonamides
(SA1–SA12) is outlined in Fig. 4. Initially, 2-acetylthiophene (I) was
brominated with bromine in dichloromethane (DCM). The key
starting compound 4-(thiophen-2-yl)thiazol-2-amine (III) was pre-
pared in a high yield by the cyclization reaction of a-bromoketones
(II) with thiourea under mild basic conditions. The combination of
the amine (III) with different benzenesulfonyl chlorides (IV) in
anhydrous pyridine at ꢀ5 1C afforded novel sulfonamides
(SA1–SA12).
Fig. 2 Schematic representation of the antibacterial activity mechanism of sulfonamides. Molecular surface representation of DHPS with the
co-crystallized PABA and DHPP (PDB code: 3TYZ)10 represented as red and black sticks, respectively.
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properties of the new sulfonamides (SA1–SA12) and commercial
derivatives (sulfathiazole and sulfadiazine) against a wide range
of bacteria (various Staphylococcus aureus strains, Listeria mono-
cytogenes, Pseudomonas aeruginosa, and Escherichia coli) and
Candida albicans, as a fungal representative, by determining
their minimal inhibitory concentrations (MICs).
According to the obtained values, the newly synthesized
sulfonamides were found to be selectively active against Staphylo-
coccus aureus strains. Besides, their effects on other bacteria and
fungi were lacking (activity above 200 mg mLꢀ1 was not considered
as a significant activity). The MIC values of SA1–SA12 against
S. aureus strains are reported in Table 2, and the complete data
against all tested microorganisms are provided in the ESI.†
Commercial sulfonamides were not significantly effective on
either bacterial or fungal strains under the tested conditions, as
expected, due to sulfonamide resistance. Among the tested
compounds, SA7, SA11 and SA12 showed the highest potency
against S. aureus strains (S. aureus ATCC 43300 (MRSA), S. aureus
ATCC 25923, S. aureus NCTC 6571, and the clinical isolate
S. aureus 861). Among these three compounds, SA7 showed the
best activity results. Against S. aureus ATCC 25923, SA7 and SA11
stood out with MIC values of 0.25 and 0.5 mg mLꢀ1, respectively.
The activities of SA7 and SA12 against MRSA were also note-
worthy with MIC values of 1 and 2.5 mg mLꢀ1, respectively.
Moreover, the compounds representing MIC values
r12.5 mg mLꢀ1 on any S. aureus strain were selected and tested
for their cytotoxic effects against the human fibroblast cell line
MRC-5. Additionally, the selectivity index (SI; a ratio that
measures the window between cytotoxicity and antimicrobial
activity by dividing the given IC50 value by the MIC value) was
determined for these compounds by selecting the best MICs
highlighted in Table 2. Cytotoxicity data as well as the best SI
values of the selected SA derivatives are presented in Table 3.
According to the obtained results, the least cytotoxicity was
determined for SA4, while the most active antibacterial SA7,
SA11 and SA12 derivatives represented IC50 values between 30
and 34 mg mLꢀ1. The SI values calculated for these compounds
Fig. 3 Representation of the molecular modifications carried out on
sulfathiazole.
Fig. 4 Reagents and conditions: (a) bromine, DCM, 0 1C to rt, 2 h; (b)
thiourea, ethanol, reflux, 2 h; (c) DCM, pyridine, ꢀ5 1C, 3 days.
In all cases, the final products (Table 1) were purified by
1
silica gel chromatography. H NMR, 13C NMR, IR, and HRMS
spectral data were used to provide a complete structural char-
acterization of SA1–SA12. SA9 was previously synthesized as
an intermediate compound by Zhang et al.,20 but its physico-
chemical properties as well as structure-elucidation data are
provided for the first time in this paper.
Bioactivity assessment
Antimicrobial activity evaluation and toxicity determination.
In this study, we presented the in vitro antimicrobial activity
Table 1 Chemical structures and some properties of the synthesized
compounds
Table 2 Minimal inhibitory concentrations (MICs, mg mLꢀ1) of SA1–SA12
against the tested S. aureus strains
Test organism: S. aureus
MRSA
S. aureus
S. aureus
Compound
ATCC 25923 ATCC 43300 NCTC 6571 861a
SA1
SA2
SA3
SA4
SA5
SA6
SA7
SA8
SA9
SA10
SA11
SA12
ST
100
12.5
12.5
12.5
50
100
0.25
12.5
25
100
0.5
6.25
4200
200
50
12.5
25
60
25
25
25
100
50
12.5
100
100
200
12.5
6.25
4200
4200
25
Compound
R
Yielda (%) m.p. (1C) Molecular weight
6.25
6.25
6.25
50
25
0.5
25
50
50
2.5
2.5
150
200
SA1
SA2
SA3
SA4
SA5
SA6
SA7
SA8
SA9
SA10
SA11
SA12
H
4-Br
4-Cl
2,5-diCl
3-NO2
4-NO2
4-I
4-CH3
4-OCH3
4-COCH3
70
28
30
50
37
51
45
33
35
48
170–172
180–182
171–173
132–134
214–216
220–222
199–201
191–193
182–184
198–200
212–214
200–202
322
400
356
390
367
367
448
336
352
364
378
399
25
100
100
1
50
100
100
6.25
2.5
4200
4200
4-C(CH3)3 40
4-C6H5 55
SD
a
a
Yields of pure isolated products after silica gel column chromatography.
Clinical isolate, ST-sulfathiazole, SD-sulfadiazine.
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Table 3 Cytotoxicity of the SA derivatives showing anti-S. aureus activity
against the human fibroblast cell line MRC-5 and the selectivity index values
SA2
SA3
SA4
SA7
SA8
SA11
SA12
IC50 (mg mLꢀ1
)
37
5.9
28
4.5
24
3.8
30
120
42
3.4
32
64
34
5.4
SIa
a
SI values in comparison to the best MIC values.
ranged from 3.4 to 120. It is worth mentioning that SA7 and
SA11 exhibited the best SI values of 120 and 64, respectively, on
S. aureus ATCC 25923. Besides, SA12 displayed a SI value of
5.4 against MRSA.
Staphylococcus aureus is a Gram-positive bacterium that
causes a wide spectrum of conditions ranging from mild skin
diseases to severe pneumonia and bacteremia. S. aureus resistance to
antibiotics has been identified and is associated with extensive
mortality.21 A large proportion of S. aureus isolates are resistant to
methicillin which is a semi-synthetic penicillin derivative of b-lactam
antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) has
also been reported to develop resistance to non-b lactam antibiotics
such as vancomycin and linezolid.22,23 Considering the increased
rate of death and the burden on the health-care system due to
S. aureus strains, it is critical to identify novel drug candidates that
are selectively effective against this bacterium.
Fig. 5 MRSA membrane potential perturbation upon exposure to SA7,
SA11, and SA12 (2ꢁ MIC values) in comparison to PBS treatment. Sample
fluorescence refers to that of the 3,30-diethyloxacarbocyanine iodide
(DiOC2(3)) dye and relates to the membrane potential as previously
shown.28 CCCP (carbonyl cyanide 3-chlorophenylhydrazone) was used
as a positive control.
Additionally, the cytotoxic effects of the newly synthesized
compounds on healthy cells can be a severe problem through-
out the discovery of novel antibacterial agents. Hence, it is of
the utmost importance that we identified new sulfonamides
with high selectivity indices in the present study.
plays a significant role in bacterial survival, it stands out as an
attractive target for antibacterial agents.27 Therefore, we measured
the potency of our active compounds to disrupt MRSA bacterial
membrane potential perturbation, which is considered a membrane-
targeting antibacterial activity mechanism. According to the obtained
data, SA7, SA11 and SA12 did not effectively perturb the plasma
membrane integrity, contrary to carbonyl cyanide m-chloro-
phenylhydrazone (CCCP), which was used as a positive control
(Fig. 5).
DNA interactions by gel electrophoresis. In many research
studies, sulfonamides and their metal complexes are reported
to bind DNA.29–31 Prompted by this information, we also
investigated DNA interactions of the most active sulfonamide
derivatives (SA7, SA11 and SA12). However, the tested compounds
did not show any interaction with double-stranded DNA which
indicates, at least for these three compounds, that bacterial DNA is
not the target for their mode of activity (Fig. S1, ESI†). In general,
metal complexes of sulfonamides are reported to bind to DNA with
stronger interactions than the ligands.32 Therefore, we surmise
that not testing our compounds as metal complexes can be the
determining factor for the lacking interaction with DNA.
By analyzing the results according to the chemical structures
of the compounds, it is obvious that introducing a thiophene ring
into the structure of sulfathiazole made a positive contribution to
the antibacterial activities of all derivatives. Additionally, it is worth
emphasizing that the most active compounds (SA7, SA11 and
SA12) carry highly lipophilic substituents at the para position
of the phenyl ring. The theoretical octanol–water partition
coefficients (log P) were calculated using Molinspiration24 for
these three derivatives. The log P values were found to be 4.30,
4.92 and 5.10 for SA7, SA11 and SA12, respectively, and significantly
increased compared to the value of 3.21 for SA1, a non-substituted
phenyl derivative. It is apparent that introducing an aromatic ring
(phenyl), a bulky alkyl group (tert-butyl) or a voluminous halogen
atom (iodine)25 on the phenyl ring increases the lipophilicity, thus
enhancing the antibacterial activity of sulfonamides. Therefore, we
conclude that the capability of sulfonamides to penetrate the
bacterial membranes of S. aureus strains due to increased lipo-
philicity is one of the factors playing a role in their activities.
Our data showed that SA7, SA11 and SA12 can kill the
pathogenic microorganisms in low amounts without any nega-
tive effect on cells, which made them favorable compounds for
further experiments.
Molecular modeling studies
Molecular docking. In order to develop binding hypotheses
of the three most active compounds, SA7, SA11 and SA12, we
Methicillin-resistant Staphylococcus aureus (MRSA) membrane performed molecular docking of the compounds into a model
potential perturbation. MRSA represents a global health problem of the PABA active site of S. aureus strain ATCC 25923 DHPS.
as a leading cause of mortality from antibiotic-resistant infections. The model was based on the X-ray crystal structure of S. aureus
Therefore, new strategies targeting MRSA are of the utmost impor- F17L/E208K double mutant DHPS in the ligand-bound confor-
tance to avert the emerging resistance.26 As the bacterial membrane mation (PDB ID: 6CLV) (Fig. 6A).33 L17 was restored to F17 using
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Fig. 6 The S. aureus DHPS active site of strain ATCC 25923, featuring the X-ray crystal structure of compound 1530, and the putative docking poses of
SA7, SA11 and SA12, along with their pharmacophores. (A) The S. aureus active site, featuring compound 1530 shown in white (PDB ID: 6CLV). The
sulfisoxazole moiety occupying the PABA active site is shown in ball and stick representation, and the pterin moiety occupying the DHPP active site is
shown as lines. The pharmacophore of the sulfa moiety of compound 1530 is shown. The E208K mutation causes a rearrangement of the R176-E179-
R204-E208 salt bridge. The protein backbone and wildtype residues are shown in grey. The DHPS F17L/E208K double mutant residues are shown in
black. K208 is not resolved in the X-ray crystal structure and is shown as A208. (B) The binding hypothesis for SA7 along with its pharmacophore. (C) The
binding hypothesis for SA11 along with its pharmacophore. (D) The binding hypothesis for SA12 along with its pharmacophore. Yellow = hydrophobic
contact. Red = hydrogen bond acceptor. Mauve = aromatic interaction.
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MOE protein builder (v2019.0102)34 (Fig. 6A and 6B), although over the course of MD simulations, delivering a visual repre-
the rotamer of F17 in the substrate-bound form of S. aureus sentation of the statistical occurrence of each 3D pharmaco-
DHPS described by Griffith et al. was not reproducible.33 This phore feature as point density clouds (Fig. 7C–E).35
may be because the model generated by Griffith et al. was based
Interestingly, the dynophores uncovered interaction features
on an X-ray crystal structure of the DHPS wildtype binding site not seen in the static 3D pharmacophores of the three com-
in Yersinia pestis (PDB ID: 3TYZ),35 which features a different pounds. In all three molecules, the sulfonamide nitrogen and
backbone geometry in the loop harboring F17. K208 was second sulfonyl oxygen act as hydrogen bond acceptors and the
restored to E208 and the R176-E179-R204-E208 four-residue salt sulfonamide moiety forms a negative ionizable feature (circled
bridge complex was modelled on X-ray coordinates of wildtype in blue in Fig. 7C–E; Table S2, ESI†). Furthermore, for SA12, the
S. aureus DHPS (PDB ID: 1AD1)35 (Fig. 6A). The F17L/E208K phenyl rings engage in aromatic interactions, the thiophene
double mutant X-ray crystal structure of DHPS from S. aureus ring forms a hydrophobic contact and its thiazole nitrogen
(PDB ID: 6CLV) features a pterin–sulfonamide hybrid com- forms a hydrogen bond (Fig. 7F and Table S2, ESI†). The SA11
pound including a sulfisoxazole moiety (compound 1530) as a thiophene ring forms an additional hydrophobic contact as
co-crystallized ligand, of which the sulfisoxazole part displaces well as aromatic interaction (Fig. 7E and Table S2, ESI†). The
the natural PABA substrate according to the classic sulfonamide SA7 iodine R-substituent forms a halogen bond, the thiazole
mechanism of action9,10 (Fig. 6A). Fig. 6B–D depict the binding ring forms an aromatic interaction, the thiazole nitrogen
pose hypotheses of SA7, SA11 and SA12, respectively, along with engages in a hydrogen bond, and the thiophene ring forms
their static pharmacophores. The suggested binding poses were an additional hydrophobic contact (Fig. 7D and Table S2, ESI†).
derived from molecular docking and filtered by 3D pharmaco-
SA11 shows the highest degree of fluctuation of ligand from
phore screening33 based on the 3D interactions of the bound the putative pose within the binding site, followed by SA12,
sulfisoxazole33 (Fig. 6A), according to the workflow detailed in with SA7 showing the least amount of movement within the
the Methods section. The poses suggest that the compounds DHPS active site (Fig. 7). The dynophores highlight the differences
demonstrate a common binding mode, with the phenyl groups in ligand–enzyme interaction patterns that may explain the
occupying the hydrophobic pocket of K203, F17, and F172, increased tendency of SA11 and SA12 to deviate from their putative
which is also occupied by the phenyl group of the natural PABA binding poses compared to SA7. The iodine as the R-substituent of
substrate.9,33 The phenyl group of SA11 engages in a further SA7 forms hydrophobic contacts to PABA active site residues 100%
pi-cation interaction with K203. A sulfonyl oxygen of each of the time, whereas the SA11 tert-butyl as the R-substituent only
compound forms a hydrogen bond with the backbone nitrogen forms hydrophobic contacts 83% of the time. The SA7 phenyl
of R204. For all three compounds, the substituents on the group engages in hydrophobic contacts with PABA active site
phenyl ring (iodine, tert-butyl and phenyl for SA7, SA11 and residues for 83% of the simulation trajectory, whereas the SA11
SA12, respectively) point towards the pterin sub-pocket, forming phenyl group only forms these contacts 63% of the simulation
hydrophobic contacts to F17 and F172. The thiazole and thio- time (Table S2, ESI†). Whereas the sulfonyl oxygens of SA7 act as
phene rings of the three compounds extend out of the natural hydrogen bond acceptors 42% and 46% of the trajectory, the
substrate envelope. Each thiazole ring engages in a lipophilic SA11 sulfonyl oxygens form hydrogen bonds 27% and 26% of
contact with F17, and the thiazole nitrogen of SA11 engages in a the simulation time (Table S2, ESI†).
hydrogen bond with R52. The SA7 and SA11 thiophene rings
The occurrence of the R-substituent hydrophobic interaction
engage in a further pi-cation interaction with R52. The thiazole is less frequent in SA12 than in SA7 (83 vs. 100%, respectively).
and thiophene rings of SA12 are rotated 1801 about the bond The aromatic interaction of the phenyl group occurs 6% of the
between the sulfonamide nitrogen and the carbon at position 2 time in SA7 and 1% of the time in SA12, and the hydrogen bond
of the thiazole ring compared to those of SA7 and SA11, thus to one sulfonyl oxygen occurs over 46% of the SA7 trajectory and
adopting the opposite orientation.
2% of the SA12 trajectory. Occurrence frequencies of 1% and 2%
Molecular dynamics simulations and dynamic 3D pharma- are almost negligible.
cophores (dynophores). In order to investigate the conforma-
The dynophore clouds of SA11 and SA12 extend into areas of
tional dynamics of SA7, SA11 and SA12 within the S. aureus the enzyme not seen in the SA7 dynophore (labelled as pockets
ATCC 25923 DHPS binding site, we performed all-atom molecular 1–3 in Fig. 7D and F) as SA11 and SA12 deviate from their original
dynamics (MD) simulations starting from the hypothesized bind- suggested binding poses to a greater extent, forming more
ing pose for each ligand (Fig. 6). As a measure of ligand mobility, interactions with residues further outside the PABA active site.
we calculated the Root Mean Square Deviation (RMSD) of the The occupancy of pocket 3 by the SA12 thiophene ring is most
ligand heavy atoms (Fig. 7A–C). The RMSD describes the deviation likely due to the difference in the orientation of this moiety in the
of the atoms from the initial suggested binding pose over the putative SA12 binding pose. We further used these dynophores to
course of the simulation. SA11 and SA12 show a higher degree of explain the activity of the three compounds at S. aureus strain
fluctuation of ligand from the putative pose within the binding site ATCC 25923.
than SA7 (Fig. 7A–C). To elucidate the molecular basis underlying
Mechanism of activity against S. aureus ATCC 25923. SA7
the mobility of the binding hypotheses within the DHPS active site, shows the highest activity at S. aureus ATCC 25923, followed by
dynamic 3D pharmacophores (dynophores) were generated. The SA11 and then SA12 (Table 2). The hydrogen bond formed by a
dynophore method tracks the ligand–protein interaction pattern sulfonyl oxygen for 46% of the SA7 trajectory occurs 26% of the
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Fig. 7 Investigations into the SA7, SA11 and SA12 binding hypotheses at the S. aureus ATCC 25923 DHPS active site via molecular dynamics simulations
and dynamic 3D pharmacophores (dynophores). RMSD values of SA7 (A), SA11 (B), and SA12 (C) over the course of five replicates of molecular dynamics
simulations. Dynophores of the SA7 (D), SA11 (E), and SA12 (F) binding hypothesis. Pockets into which the SA7, SA11 and SA12 dynophores spread are
circled in black and labelled with the pocket name. Pocket 1 is defined by residues I9, M128, F172 and A199. Pocket 2 is defined by F17, S50, T51 and R52.
Pocket 3 is defined by N11, T13, F17, R204, T215, P216 and R219. Negative ionizable feature clouds are circled in blue. Yellow cloud = hydrophobic
contact. Blue cloud = aromatic interaction. Red cloud = Hydrogen bond acceptor or negative ionizable feature.
SA11 simulation time, and 2% of the SA12 trajectory, which is could also contribute to the decreased activity of SA12 at S.
almost negligible. As the occurrence frequency of this hydrogen aureus ATCC 25923 compared to the other two compounds.
bond decreases with decreasing compound activity at S. aureus Additionally, the reduced potency of SA11 and SA12 compared to
ATCC 25923, it could be an underlying cause of this activity SA7 (Table 2) could be explained by the fact that SA11 and SA12
trend. Furthermore, the initial binding hypotheses show the are not able to maintain the rigidity of the binding pose within
SA12 thiazole and thiophene moieties oriented oppositely to the PABA active site associated with the higher activity of SA7.
those of SA7 and SA11, and SA12 displays the fewest number of
static modeled interactions. Dynophore analysis shows that
SA12 is able to recapitulate neither the aromatic interaction
of the thiazole ring displayed by SA7 and SA11 nor the aromatic
interaction formed by the SA-11 thiophene moiety. The SA12
Experimental
Chemistry
thiophene also displays the lowest hydrophobic contact occur-
Materials and methods. All reagents were purchased from
rence frequency (Table S2, ESI†). This difference in putative commercial sources and used as received. Melting points were
binding poses and the resulting dynophore interaction patterns determined using open glass capillaries and the results are
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uncorrected. Infrared (IR) spectra were obtained in the range of
4-Chloro-N-(4-(thiophen-2-yl)thiazol-2-yl)benzenesulfonamide
4000–600 cmꢀ1 via ATR diamond. H-(400 MHz) and 13C-NMR (SA3). Yellow solid, Rf (40: 60 EtOAc : hexane): 0.36; IR (ATR)
spectra (100 MHz) were recorded using a Bruker AM 400 3246, 3174, 1523, 1180. 1H NMR (400 MHz, DMSO-d6) d 13.46 (bs,
spectrometer in DMSO-d6 solution. Coupling constants, J, are 1H, –NH), 7.84 (bs, 2H), 7.64 (bd, J = 7.9 Hz, 3H), 7.50 (bs, 1H),
reported in hertz. MS spectra were measured on an Agilent 7.12 (d, J = 4.3 Hz, 1H), 7.05 (s, 1H). 13C NMR (100 MHz, DMSO-
1200/6530 LC/MS High-Resolution Time of Flight (TOF) instru- d6) d 168.97, 141.23, 137.55, 131.70, 129.69, 128.60, 128.27,
ment at the Atatu¨rk University-East Anatolian High Technology 128.18, 127.89, 126.51, 103.29. HRMS (EI): [M + H]+, found
1
Research and Application Center (DAYTAM).
356.9571. C13H9ClN2O2S3 requires 356.9593.
Synthesis of 2-(bromoacetyl)thiophene (II). Following the
2,5-Dichloro-N-(4-(thiophen-2-yl)thiazol-2-yl)benzenesulfon-
method reported in the literature,37 a solution of bromine amide (SA4). Pink solid, Rf (50 : 50 : 1 EtOAc : hexane : MeOH):
(0.81 mL, 15.85 mmol) in CH2Cl2 (5 mL) was added dropwise 0.30; IR (ATR) 3174, 3105, 1527, 1134. 1H NMR (400 MHz,
to a solution of 2-acetylthiophene (15.85 mmol) in CH2Cl2 DMSO-d6) d 13.61 (bs, 1H, –NH), 8.04 (d, J = 2.4 Hz, 1H), 7.76–
(5 mL) at 0 1C. The reaction mixture was stirred at room 7.66 (m, 2H), 7.64 (d, J = 5.1 Hz, 1H), 7.54 (d, J = 3.9 Hz, 1H),
temperature for 2 h, neutralized with a saturated solution of 7.15–7.11 (m, 1H), 7.06 (s, 1H). 13C NMR (100 MHz, DMSO-d6) d
sodium hydrogen carbonate and extracted with CH2Cl2 (3 ꢁ 25 mL). 169.69, 141.12, 134.00, 133.77, 132.27, 131.84, 131.47, 130.34,
The combined CH2Cl2 layers were then dried over anhydrous 129.55, 128.58, 127.96, 126.69, 103.71. HRMS (EI): [M + H]+,
MgSO4 and removed under reduced pressure. A pale yellow oil found 390.9178. C13H8Cl2N2O2S3 requires 390.9203.
product was used for the next step without purification.
3-Nitro-N-(4-(thiophen-2-yl)thiazol-2-yl)benzenesulfonamide
Synthesis of 4-(thiophen-2-yl)thiazol-2-amine (III). Following (SA5). Yellow solid, Rf (50 : 50 : 1 EtOAc : hexane : MeOH): 0.35;
the method reported in the literature,38,39 a solution of 2-bromo- IR (ATR) 3211, 3105, 1537, 1137. 1H NMR (400 MHz, DMSO-d6)
acetylthiophene (5.13 g, 0.025 mol) and thiourea (0.03 mol) in d 13.60 (bs, 1H, –NH), 8.51 (s, 1H), 8.44 (d, J = 8.5 Hz, 1H), 8.26
ethanol (20 mL) were stirred for 15 min then refluxed for 2 h. (d, J = 8.0 Hz, 1H), 7.87 (t, J = 8.2 Hz, 1H), 7.61 (d, J = 5.1 Hz, 1H),
Upon cooling, the reaction mixture was made alkaline using 7.50 (d, J = 3.8 Hz, 1H), 7.15–7.09 (m, 1H), 7.05 (s, 1H). 13C NMR
10% sodium hydroxide solution with stirring. The precipitate (100 MHz, DMSO-d6) d 169.47, 148.20, 144.17, 132.54, 132.32,
was collected, washed with water and dried. Recrystallization 131.77, 131.67, 128.56, 127.88, 127.27, 126.54, 120.87, 103.53.
with ethanol afforded the desired compound. Mp 124–126 1C; HRMS (EI): [M + H]+, found 367.9812. C13H9N3O4S3 requires
1H NMR (400 MHz, DMSO-d6) d 7.37 (dd, J = 8.0, 4.2 Hz, 1H), 367.9833.
7.17 (s, 2H), 7.13–6.95 (m, 1H), 6.83 (s, 1H). 13C NMR (100 MHz,
4-Nitro-N-(4-(thiophen-2-yl)thiazol-2-yl)benzenesulfonamide
DMSO) d 168.72, 144.90, 139.62, 128.19, 125.14, 123.19, 100.22. (SA6). Yellow solid, Rf (60 : 40 : 1 EtOAc : hexane : MeOH): 0.32;
HRMS (EI): [M + H]+, found 183.00452. C7H6N2S2 requires IR (ATR) 3263, 3122, 1517, 1137. 1H NMR (400 MHz, DMSO-d6)
183.00694.
d 13.46 (bs, 1H, –NH), 8.37 (d, J = 8.3 Hz, 2H), 8.08 (d, J = 8.3 Hz,
General procedure for the synthesis of sulfonamides SA1–SA12. 2H), 7.62 (d, J = 5.0 Hz, 1H), 7.50 (d, J = 3.8 Hz, 1H), 7.12 (d, J =
A solution of 4-(thiophen-2-yl)thiazol-2-amine 0.15 g (0.81 mmol) 4.8 Hz, 1H), 7.06 (d, J = 2.7 Hz, 1H). 13C NMR (100 MHz, DMSO-
and benzenesulfonyl chloride derivatives (1.2 mmol) in dichloro- d6) d 169.69, 149.81, 147.99, 128.59, 127.92, 127.81, 126.51,
methane and pyridine (15 mL, 12 : 3) were stirred at ꢀ5 1C for three 124.97, 124.89, 103.50. (One signal was overlapped). HRMS (EI):
days (completion of the reaction was monitored by TLC). After the [M + H]+, found 367.9807. C13H9N3O4S3 requires 367.9833.
reaction was completed, the solvent was evaporated in vacuum.
4-Iodo-N-(4-(thiophen-2-yl)thiazol-2-yl)benzenesulfonamide
The solid residue was purified by column chromatography. (SA7). White solid, Rf (60 : 40 : 1 EtOAc : hexane: MeOH): 0.26; IR
1
Suitable solvent washing and crystallization methods were pre- (ATR) 3193, 3070, 1531, 1134. H NMR (400 MHz, DMSO-d6) d
ferred to obtain the pure product.
13.34 (bs, 1H, –NH), 7.93 (d, J = 8.3 Hz, 2H), 7.60 (d, J = 7.9 Hz,
N-(4-(Thiophen-2-yl)thiazol-2-yl)benzenesulfonamide (SA1). 3H), 7.55–7.45 (m, 1H), 7.13–7.05 (m, 1H), 7.01 (bs, 1H). 13C
White solid, Rf (40 : 60 EtOAc : hexane): 0.22; IR (ATR) 3157, NMR (100 MHz, DMSO-d6) d 168.91, 142.10, 138.38, 132.35,
3087, 1517, 1147. 1H NMR (400 MHz, DMSO-d6) d 13.34 (bs, 1H, 131.95, 128.54, 128.10, 127.75, 126.41, 103.24, 100.48. HRMS
–NH), 7.84 (d, J = 7.6 Hz, 2H), 7.71–7.50 (m, 4H), 7.49 (d, J = 3.7 (EI): [M + H]+, found 448.8930. C13H9IN2O2S3 requires 448.8949.
Hz, 1H), 7.13–7.07 (m, 1H), 7.02 (bs, 1H). 13C NMR (100 MHz,
4-Methyl-N-(4-(thiophen-2-yl)thiazol-2-yl)benzenesulfonamide
DMSO-d6) d 148.40, 146.68, 142.40, 129.54, 129.07, 128.19, (SA8). Grey solid, Rf (50 : 50 : 1 EtOAc : hexane: MeOH): 0.43; IR
1
128.13, 127.72, 126.31, 125.93, 103.27. HRMS (EI): [M + H]+, (ATR) 3166, 3087, 1527, 1137. H NMR (400 MHz, DMSO-d6) d
found 322.9978. C13H10N2O2S3 requires 322.9983.
13.22 (s, 1H), 7.74 (d, J = 7.9 Hz, 2H), 7.60 (d, J = 5.1 Hz, 1H), 7.49
4-Bromo-N-(4-(thiophen-2-yl)thiazol-2-yl)benzenesulfonamide (d, J = 3.7 Hz, 1H), 7.36 (d, J = 7.7 Hz, 2H), 7.11 (t, J = 4.5 Hz, 1H),
(SA2). White solid, Rf (40 : 60: 1 EtOAc : hexane: MeOH): 0.37; IR 7.01 (s, 1H), 2.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d 168.25,
1
(ATR) 3246, 3105, 1527, 1134. H NMR (400 MHz, DMSO-d6) d 143.03, 139.47, 132.88, 132.21, 129.91, 128.53, 127.61, 126.45,
13.38 (bs, 1H, –NH), 7.77 (s, 4H), 7.61 (d, J = 5.2 Hz, 1H), 7.49 126.27, 103.12, 21.41. HRMS (EI): [M + H]+, found 337.0118.
(d, J = 3.8 Hz, 1H), 7.15–7.08 (m, 1H), 7.03 (s, 1H). 13C NMR C14H12N2O2S3 requires 337.0139.
(100 MHz, DMSO-d6) d 169.03, 141.70, 132.59, 131.76, 128.56,
4-Methoxy-N-(4-(thiophen-2-yl)thiazol-2-yl)benzenesulfonamide
128.37, 127.83, 126.51, 126.44, 123.22, 103.28. HRMS (EI): (SA9). White solid, Rf (40 : 60 EtOAc : hexane): 0.35; IR (ATR)
1
[M + H]+, found 400.9081. C13H9BrN2O2S3 requires 400.9088.
3174, 3018, 1537, 1124. H NMR (400 MHz, DMSO-d6) d 13.23
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(bs, 1H, –NH), 7.77 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 5.1 Hz, 1H), bromide (MTT) was performed using compounds with MICs of
7.48 (d, J = 3.8 Hz, 1H), 7.16–6.87 (m, 4H), 3.80 (s, 3H). 13C NMR up to 50 mg mLꢀ1 41
Cells were seeded at a concentration of
.
(100 MHz, DMSO-d6) d 172.56, 162.61, 134.04, 131.88, 128.71, 1 ꢁ 104 cells per well in RPMI 1640 medium supplemented with
128.57, 127.64, 126.24, 114.71, 114.65, 103.21, 56.05. HRMS (EI): 100 mg mLꢀ1 streptomycin, 100 U mLꢀ1 penicillin, and 10% (v/v)
[M + H]+, found 353.0067. C14H12N2O3S3 requires 353.0088.
FBS. Cells were grown in an atmosphere of 95% air and 5% CO2
4-Acetyl-N-(4-(thiophen-2-yl)thiazol-2-yl)benzenesulfonamide at 37 1C and their viability was measured after 48 h. The scope of
(SA10). Yellow solid, Rf (50 : 50 : 1 EtOAc : hexane : MeOH): 0.28; MTT reduction was measured using the Tecan Infinite 200 Pro
IR (ATR) 3246, 3105, 1537, 1134. 1H NMR (400 MHz, DMSO-d6) d multiplate reader (Tecan Group Ltd, Mannedorf, Switzerland) by
¨
13.41 (bs, 1H, –NH), 8.11 (d, J = 8.3 Hz, 2H), 7.97 (d, J = 8.1 Hz, measuring the absorbance at 540 nm. The concentrations of
2H), 7.61 (d, J = 5.1 Hz, 1H), 7.50 (d, J = 3.8 Hz, 1H), 7.11 (t, J = compounds which were able to inhibit cell growth by 50% (IC50)
4.4 Hz, 1H), 7.04 (s, 1H), 2.61 (s, 3H). 13C NMR (100 MHz, are presented as cytotoxicity indicators.
DMSO-d6) d 197.76, 169.06, 146.06, 139.75, 132.44, 131.81,
Staphylococcus aureus MRSA membrane potential perturba-
129.37, 128.56, 127.83, 126.65, 126.49, 103.34, 27.43. HRMS tion. The depolarization of the membrane potential of S. aureus
(EI): [M + H]+, found 365.0058. C15H12N2O3S3 requires 365.0088. MRSA was determined as described previously.28 S. aureus
4-(Tert-butyl)-N-(4-(thiophen-2-yl)thiazol-2-yl)benzenesulfon- MRSA was grown in LB medium to the OD625nm of 0.3, and
amide (SA11). White solid, Rf (60 : 40 : 1 EtOAc : hexane : MeOH): after it was washed with PBS (pH 7.4), 1 ꢁ 108 CFU mLꢀ1 were
0.25; IR (ATR) 3263, 3122, 1537, 1141. 1H NMR (400 MHz, resuspended in PBS containing 1% (w/v) glucose. The resuspended
DMSO-d6) d 13.07 (bs, 1H, –NH), 7.77 (d, J = 8.0 Hz, 2H), bacterial cells were incubated for 15 min in a 37 1C shaker
7.58 (t, J = 8.0 Hz, 3H), 7.48 (d, J = 3.8 Hz, 1H), 7.14–7.06 (m, (180 rpm), followed by incubation with the 30-diethyloxacarbo-
1H), 7.02 (d, J = 9.4 Hz, 1H), 1.27 (s, 9H). 13C NMR (100 MHz, cyanine iodide (DiOC2(3)) dye (final concentration of 3 mM) for
DMSO-d6) d 168.48, 155.81, 139.50, 132.70, 132.14, 128.56, 30 min in a 37 1C shaker (180 rpm). After the second incubation
127.70, 127.01, 126.34, 126.27, 103.20, 35.26, 31.25. HRMS with the dye, bacterial cells were transferred to a 96 well plate
(EI): [M + H]+, found 379.0593. C16H17N2O2S3 requires 379.0609. (100 mL per well) and treated with different sulfonamide com-
N-(4-(Thiophen-2-yl)thiazol-2-yl)-[1,10-biphenyl]-4-sulfonamide pounds. The compounds were prepared in a microtiter plate at
(SA12). White solid, Rf (60 : 40 : 1 EtOAc : hexane: MeOH): 0.25; IR concentrations of 0.5ꢁ MIC, 1ꢁ MIC, and 2ꢁ MIC. As a negative
1
(ATR) 3263, 3122, 1537, 1180. H NMR (400 MHz, DMSO-d6) d control, PBS with 1% (w/v) glucose was used, and carbonyl
13.36 (bs, 1H, –NH), 7.93 (m, 7.96–7.89, 2H), 7.90–7.80 (m, 2H), cyanide m-chlorophenyl hydrazone (CCCP) as a positive control
7.73 (t, J = 6.3 Hz, 2H), 7.65–7.57 (m, 1H), 7.56–7.30 (m, 4H), (final concentration of 0.025 mM). Immediately after the addition
7.19–6.93 (m, 2H). 13C NMR (100 MHz, DMSO-d6) d 168.65, of compounds, using a Tecan Infinite 200 Pro multiplate reader
144.35, 141.14, 139.09, 132.02, 129.55, 128.87, 128.55, 128.55, (Tecan Group Ltd, Mannedorf, Switzerland), the fluorescence
127.76, 127.73, 127.49, 127.02, 126.40, 103.24. HRMS (EI): change was measured (excitation 485 nm, emission 630 nm).
[M + H]+, found 399.0268. C19H14N2O2S3 requires 399.0296.
DNA interactions by gel electrophoresis. The DNA inter-
action assay was performed using the commercial lambda
bacteriophage DNA (300 ng, Thermo Scientifict) according to
Biological evaluation
Antimicrobial assays. Stock solutions of sulfonamides SA1–SA12 the previously published procedure.42 20 ng mLꢀ1 of DNA
were prepared in DMSO at a final concentration of 50 mg mLꢀ1 and solution was incubated with 400 mM, 200 mM, and 100 mM of
kept at 4 1C before use, no more than two weeks. Minimal inhibitory compounds in a 15 mL reaction volume at 37 1C for 1 h. After the
concentration (MIC) values against Staphylococcus aureus ATCC period of incubation, the samples were mixed with a loading
25923, Staphylococcus aureus NCTC 6571, Staphylococcus aureus dye and run on 0.8% (w/v) agarose gel containing ethidium
ATCC43300 (MRSA), Staphylococcus aureus isolate 861 (veterinary bromide (EtBr) at 60 V for 1 h. HyperLaddert 1 Kb Plus DNA
clinical isolate, Vetlab Ltd, Belgrade, Serbia), Listeria monocytogenes Ladder (FastGene) was used as standart. Results were obtained
NCTC 11994, Pseudomonas aeruginosa NCTC 10332, Escherichia coli using the Gel Doc EZ system (Bio-Rad, Life Sciences, Hercules,
ATCC 11775, and Candida albicans ATCC 10231 were determined in USA), equipped with the Image Labt software.
Luria-Bertani broth (10.0 g Lꢀ1 tryptone, 10.0 g Lꢀ1 NaCl, 5.0 g Lꢀ1
Molecular modeling studies
yeast extract, pH 7.2) and RPMI 1640 medium (Gibco) according to
the standard broth microdilution assays.40 The concentration
Molecular docking. The X-ray coordinates of double mutant
of inoculum was 5 ꢁ 105 CFU mLꢀ1 for bacterial and 1 ꢁ ligand-bound DHPS from S. aureus (PDB ID: 6CLV)33 were taken
106 CFU mLꢀ1 for fungal cells and the maximum tested as the template protein conformation and prepared and restored
compound concentration was 200 mg mLꢀ1. The MIC values of to wildtype in the MOE protein builder (v2019.0102).34 The
growth were obtained by measuring the absorbance at 600 nm mutant residues F17L and E208K were restored to wildtype
(OD600) after overnight incubation at 37 1C using a Tecan Infinite and energy-minimized using the OPLS-AA forcefield,43 and the
¨
200 Pro multiplate reader (Tecan Group Ltd, Mannedorf, Switzer- R176-E179-R204-E208 salt bridge conformation was modelled
land). DMSO as a vehicle solvent was used as a control. manually according to the X-ray coordinates of wildtype
Antiproliferative activity assay on a human fibroblast cell line. S. aureus DHPS in the apo form (PDB ID: 1AD1). The unresolved
On a human lung fibroblast cell line (MRC-5), an antiproliferative 6-residue loop D19-N24 was modelled using the loop modeller
activity assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium function in MOE. The resulting models had no geometric outliers.
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The enzyme structures were protonated at pH 7.4 using the feature. The resulting so-called ‘superfeatures’ are then
Protonate3D.44 The 3D structures of compounds SA7, SA11 represented as density clouds (Fig. 7D–F) according to their
and SA12 were generated using ChemDraw (v18.1; PerkinElmer statistical occurrence throughout the course of the MD simulation
Informatics) and Corina (v3.0; Molecular Networks GmbH).45
and which protein residues are involved. Dynophores offer a deeper
Compounds SA7, SA11 and SA12 were flexibly docked into analysis of protein–ligand interaction dynamics, for example, to
the rigid modelled wildtype structure of DHPS from S. aureus explain the effect of resistance mutations on CYP4Z1 substrate
using GOLD (v.5.2; Genetic Optimization for Ligand Docking, recognition.55 Dynophores have provided insights into the plasticity
The Cambridge Crystallographic Data Centre, UK).46 The sulfonyl of the arginase catalytic site, resulting in the development of two
sulphur was used as the center of the binding site sphere and the arginase inhibitors, which hold the potential to inform the devel-
radius was set to 10 Å. The genetic algorithm (GA) performed opment of anticancer therapeutics.56 Dynophores have also been
50 runs at 100% efficiency and was set to generate diverse solutions. used to explain an activity cliff between two M2 receptor ligands36
The resulting 50 binding hypotheses were energy-minimized using and to uncover a mechanism of bypassing drug resistance in HIV-
the MMFF94 forcefield47 in LigandScout (v 4.4, Inte:Ligand, Vienna, reverse transcriptase.57 The dynophore method is fully automated
Austria)48–50 in the presence of the wildtype S. aureus DHPS model. and implemented in the LigandScout framework.48–50
The poses were filtered according to how well they fulfil the
features of the 3D pharmacophore model of the sulfa moiety of
the compound co-crystallized in the F17L/E208K DHPS X-ray
Conclusion
crystal structure (PDB ID: 6CLV)33 (Fig. 6A). Compounds in
which the phenyl group did not overlap with the phenyl group The discovery of sulfonamides as the first class of synthetic
of the co-crystallized ligand in the hydrophobic pocket of the antibacterial agents initiated the golden era in the management
PABA catalytic site were rejected. Only binding modes wherein of bacterial infections. With their wide spectrum of antibacterial
a sulfonyl oxygen forms hydrogen bonds with the backbone activities, sulfonamides were successfully used for more than
nitrogen of R204 were retained. Binding hypotheses were 75 years to control bacterial infections until the development of
further filtered by the number of interactions with the protein prevalent drug resistance. In the present work, we aimed
and by the fit of the compound into the PABA active site.
to overcome this sulfonamide resistance through rational mole-
Molecular dynamics simulations. The complexes of S. aureus in cular modifications. Therefore, we synthesized twelve new
complex with SA7, SA11, SA12 and the co-crystallized ligand were modified sulfonamide derivatives based on the structure of
prepared in Maestro (v11.7),51 and 5 replicates of unrestrained sulfathiazole, a well-known antibacterial sulfa drug. Introducing
molecular dynamics simulation of each system were performed a thiophene ring into the main scaffold and lipophilic substi-
using Desmond (v. 5.5)52 and the OPLS-AA forcefield.43 The tuents at the para position of the phenyl ring led to a significant
simulations were performed on water-cooled Nvidia GeForce increase in the antibacterial activities of the compounds against
RTX 2080 Ti graphics cards (NVIDIA corporation, Santa Clara, S. aureus. Three compounds (SA7, SA11 and SA12) represent the
USA). Systems were solvated in a cubic water box with 15 Å most attractive ones in this series with low MIC values and low
padding to the protein surface using TIP4P water molecules.53 cytotoxicity against healthy human lung fibroblasts as well. The
Sodium ions were added to neutralize the system, and NaCl was MRSA membrane potential perturbation and DNA interaction
added to a physiological concentration of 0.15 M. Simulations characteristics of SA7, SA11 and SA12 indicated that these
were run for 50 ns, and 1000 ligand–complex conformations mechanisms are unlikely to play a role in the antibacterial
were obtained for each replicate. Simulations were performed at activity of these compounds. Molecular docking of SA7, SA11
a constant pressure of 1.01325 bar using the Martyna–Tobias– and SA12 into the S. aureus ATCC 25923 DHPS active site shows
Klein method and a temperature of 300 K using the Nose– a commonly hypothesized binding mode for SA7 and SA11, with
Hoover chain method. Intermolecular forces were calculated the thiazole and thiophene rings of SA12 adopting a different
using the RESPA integrator with a standard interaction cutoff of conformation to the former two compounds. Thiophene ring
9.0 Å. VMD (v1.9.3)54 was used for the visual inspection of the and lipophilic substituents (iodine, tert-butyl and phenyl) pro-
conformational trajectory of the ligands and for the calculation vided additional aromatic and hydrophobic interactions with
of RMSD values.
DHPS. Molecular dynamics simulations underline that SA7 is
Dynamic 3D pharmacophores (dynophores). The dynophore able to retain its original suggested binding pose to a higher
approach combines static 3D pharmacophore modelling with degree than SA11 and SA12, in line with its increased potency.
conformational sampling of the ligand–enzyme complex This difference in ligand rigidity within the active site could be
through molecular dynamics (MD) simulations.36 The five MD explained by dynamic 3D pharmacophore analysis, which
simulation replicates of each ligand–enzyme system were elucidated the differences in ligand–enzyme interaction patterns
concatenated and aligned to the heavy atoms of the starting between the three compounds. The activity trend followed by the
conformation. A 3D pharmacophore model was generated for compounds at S. aureus ATCC 25923 could be traced back to the
each enzyme–ligand conformation generated during the MD occurrence frequency of a hydrogen bond formed by a sulfonyl
simulations. Interaction features are categorized as aromatic, oxygen. Altogether, these data suggest a new approach to modify
hydrophobic, charged interactions, or hydrogen bonds, and are sulfa drugs to overcome sulfonamide resistance and improve the
grouped together according to the ligand atoms involved in antibacterial activity, particularly against S. aureus strains.
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Conflicts of interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgements
-S. D. D. and Z. K. are grateful to the Research Foundation of
Erciyes University (Project Number: FBA-2017-7340) for the
financial support of this work. We thank the Sonnenfeld
Foundation for the financial support to T. N., S. S. B., S. V.
and J. N. R. are partially supported by Ministry of Education,
Science and Technological Development of the Republic of
Serbia, 451-03-9/2021-14/ 200042.
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